Decaprenyl diphosphate synthetase gene

ABSTRACT

The present invention provides a prenyl diphosphate synthetase and a gene coding for the synthetase. The invention discloses a recombinant protein having the amino acid sequence shown in SEQ ID NO:2 or a recombinant protein which has the amino acid sequence shown in SEQ ID NO:2 having deletion, substitution or addition of at least one amino acid and which has decaprenyl diphosphate synthetase activity; a gene coding for the protein; a recombinant vector comprising the gene; a transformant transformed with the vector; a method for producing a decaprenyl diphosphate synthetase; and a method for producing ubiquinone-10.

This is a division of U.S. patent application Ser. No. 09/025,819, filedFeb. 19, 1998, now U.S. Pat. No. 6,225,097, which is incorporated hereinby reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a decaprenyl diphosphate synthetase, agene coding for the synthetase, a recombinant vector comprising thegene, a transformant transformed with the vector, a method for producinga decaprenyl diphosphate synthetase, and a method for producingubiquinone-10.

2. Description of the Prior Art

Isoprenoids are the most varied group of compounds including more than23,000 species occurring in nature. They include sterols, carotenoids,sugar carrier lipids, prenyl quinones, prenylated proteins, etc. (FIG.1). Those enzymes which catalyze the formation of carbon skeletons thatwill be the basis for the biosynthesis of these isoprenoid compounds(i.e., enzymes which catalyze the head-to-tail type condensationpolymerization of isopentenyl diphosphate (IPP) that is 5-carbonisoprene unit) are generically called as prenyl diphosphate synthetase.Prenyl diphosphate synthetase is classified into 4 groups depeding onthe chain length, conformation, etc. of the prenyl diphosphate generated(Table 1).

TABLE 1 Classification of Prenyltransferase Major Group StructureCharacteristic Products Short-chain prenyl Homodimer Soluble C₁₆, C₂₀diphosphate synthetase Medium-chain prenyl Heterodimer Soluble C₃₀, C₃₅diphosphate synthetase (E)-polyprenyl diphosphate Homodimer Activated byC₄₀, C₄₅, synthetase carrier proteins. C₅₀ (Z)-polyprenyl diphosphateHomodimer Activated by C₄₅, C₅₅ synthetase lipids.

Short-chain prenyl diphosphate synthetase (prenyltransferase I) includesgeranyl diphosphate (GPP, C10) synthetase, farnesyl diphosphate (FPP,C15) synthetase (Eberhardt, N. L. et al., (1975) J. Biol. Chem.250:863-866), geranylgeranyl diphosphate (GGPP, C20). synthetase(Sagami, H. et al. (1994) J. Biol. Chem. 269:20561-20566) and the like.The short-chain prenyl diphosphates biosynthesized by these enzymes arewater-soluble. They may be supplied as an allyl primer substrate forpolyprenyl diphosphate synthetase belonging to other groups.

Medium-chain prenyl diphosphate synthetase (prenyltransferase II)includes hexaprenyl diphosphate (HexPP, C30) synthetase (Fujii, H. etal., (1982) J. Biol. Chem., 257:14610), heptaprenyl diphosphate (HepPP,C35) synthetase (Takahashi, I. et al., (1980) J. Biol Chem., 255: 4539)and the like. These enzymes are greatly different from the short-chainprenyl diphosphate synthetase described above in that they areheterodimeric enzymes composed of two proteins each of which does nothave a catalytic function alone. Usually, these two proteins aredissociated, but when a substrate is present, they associate with eachother to manifest a function as an enzyme. Although those productsproduced by such enzymes are highly hydrophobic and apt to formmicelles, they do not require lipids nor surfactants for themanifestation of their enzyme activity. This is considered due to thefact that the medium-chain prenyl diphosphate synthetase is a specialsystem in which such dynamic dissociation and association are repeated.

E-type long-chain prenyl diphosphate synthetase (prenyltransferase III)includes octaprenyl diphosphate (OctPP, C40) synthetase, decaprenyldiphosphate (DPP, C50) synthetase and the like. Unlike prenyltransferaseII, these enzymes are undissociable homodimers and activated bypolyprenyl diphosphate carrier proteins (Ohnuma, S. et al., (1991) J.Biol. Chem. 266:23706-23713). This activation is believed to maintainthe catalyst turnover by removing hydrophobic reaction products from theactive sites of these enzymes.

Z-type long-chain prenyl diphosphate synthetase (prenyltransferase IV)includes nonaprenyl diphosphate (E,E-farnesyl-all-Z-hexaprenyldiphosphate, C45) synthetase, undecaprenyl diphosphate(E,E-farnesyl-all-Z-octaprenyl diphosphate, C55) synthetase and thelike. Reaction products generated by these enzymes work as sugar carrierlipids in the biosynthesis of bacterial cell walls. These enzymes needthe addition of a phospholipid or surfactant for the manifestation oftheir activity. DPP synthetase, which is classified intoprenyltransferase III, is also known to require a surfactant for themanifestation of its enzyme activity.

A soil bacterium Paracoccus denitrificans is a bacterium which isbelieved to be the origin of human mitochondria. The respiratory chainand the oxidative phosphorylation mechanism of this bacterium are moreefficient and more united as one organization than those of otherbacteria. Thus, the characteristics of P. denitrificans are more closerto those of mitochondria (John, P. et al., (1975) Nature, 254, 495-498).Three types of prenyl diphosphate synthetase activities have beenconfirmed from P. denitrificans (FIG. 2). They are activities of (i) FPPsynthetase which catalyzes E-type condensation of dimethylallyldiphosphate (DMAPP) with 2 molecules of IPP to produce FPP; (ii)nonaprenyl diphosphate (NPP) synthetase which catalyzes Z-typecondensation of FPP with 6 molecules of IPP to produce NPP (Ishii, K. etal., (1986) Biochem. J., 233, 773-777); and (iii) DPP synthetase whichcatalyzes E-type condensation of FPP with 7 molecules of IPP to produceDPP (Ishii K. et al., (1983) Biochem. Biophys. Res. Commun., 116,500-506).

NPP produced by NPP synthetase becomes a sugar carrier lipid which isessential for the biosynthesis of the cell wall of this bacterium.However, unlike several E-type prenyl diphosphate synthetases which havebeen already cloned and analyzed, prenyl diphosphate synthetases such asNPP synthetase and undecaprenyl diphosphate synthetase which catalyzeZ-type condensation reaction have not been elucidated yet inrelationships between their structures and enzymatic functions.

DPP produced by DPP synthetase is metabolized on the prenyl side chainof ubiquinone-10 (a constituent of the electron transport system)produced by this bacterium. All of the C30-C50 polyprenyl diphosphatesbiosynthesized by bacterial prenyltransferase II or III are provided asa side chain precursor of the corresponding menaquinone or ubiquinone.Therefore, the chain length of the product of each enzyme is directlyreflected in the side chain length of the prenylquinone of the bacteriumfrom which the enzyme is derived. Among prenylquinones, ubiquinone-10 isindustrially extracted from Paracoccus denitrificans and used aspharmaceuticals since it has the same side chain length as that of humancoenzyme Q (CoQ). Ubiquinone has been known to be effective for chronicheart diseases (Yamamura, T. (1977) Clinical Status of Coenzyme Q andProspects 281-298). Ubiquinone-10 is also effective as an antiarrhythmicagent and, thus, is utilized for the prevention of arrhythmia and thelike (Fujioka, T. et al. (1983) Tohoku J. Exp. Med. 141, 453-463).

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a decaprenyldiphosphate synthetase, a gene coding for the synthetase, a recombinantvector comprising the gene, a transformant transformed with the vector,a method for producing the decaprenyl diphosphate synthetase, and amethod for producing ubiquinone-10.

As a result of intensive and extensive researches toward the solution ofthe above assignment, the present inventor has succeeded in cloning agene coding for a long-chain decaprenyl diphosphate synthetase fromParacoccus denitrificans. Thus, the present invention has been achieved.

The present invention relates to a recombinant protein (a) or (b)described below:

(a) a protein having the amino acid sequence shown in SEQ ID NO:2

(b) a protein which has the amino acid sequence shown in SEQ ID NO:2having deletion, substitution or addition of at least one amino acid andwhich has decaprenyl diphosphate synthetase activity.

The present invention also relates to a gene coding for the recombinantprotein (a) or (b) described above. Specific examples of this geneinclude a gene comprising the base sequence shown in SEQ ID NO: 1.

Further, the present invention relates to a recombinant vectorcomprising the above gene.

The present invention further relates to a transformant transformed withthe above vector.

The present invention further relates to a method for producing adecaprenyl diphosphate synthetase comprising culturing the abovetransformant in a medium and recovering a decaprenyl diphosphatesynthetase from the resultant culture.

The present invention further relates to a method for producingubiquinone-10comprising extracting ubiquinone-10 from the abovetransformant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the biosynthesis of isoprenoid compounds.

FIG. 2 is a diagram showing the biosynthetic pathway of prenyldiphosphates in P. denitrificans.

FIG. 3 is a diagram showing the design of PCR primers.

FIG. 4 is a photograph showing the results of PCR.

FIG. 5 is a diagram showing comparison of amino acid homology.

FIG. 6 is a diagram showing the design of PCR primers.

FIGS. 7A and 7B provide two electrophorograms showing the results ofSouthern hybridization.

FIG. 8 is an electrophorogram showing the results of Southernhybridization.

FIG. 9 is an electrophorogram showing the results of Southernhybridization.

FIG. 10 is a diagram showing the structure of plasmid p11A1.

FIG. 11 is a diagram showing the open reading frame contained in plasmidp11A1.

FIG. 12 is a diagram showing comparison of amino acid sequences forvarious prenyltransferases.

FIG. 13 is an illustrative diagram showing genes located upstream anddownstream of the gene of the present invention.

FIG. 14 provides photographs of reversed phase thin layer liquidchromatograms.

FIG. 15 is a photograph showing the results of SDS-polyacrylamide gelelectrophoresis.

FIG. 16 provides HPLC charts showing the results of analysis of quinoneside chains.

FIG. 17 is a graph showing the ratios of ubiquinone production inindividual microorganisms.

DETAILED DESCRIPTION OF THE INVENTION

Hereinbelow, the present invention will be described in detail.

It is known that prenyl diphosphate synthetases (hereinafter, sometimesreferred to as “prenyltransferase(s)”) have 7 regions which have beenpreserved highly beyond species (Koyama, T. et al., (1993) J. Biochem.113:355). In the present invention, degenerate oligonucleotides for useas primers are designed based on the amino acid sequences highlypreserved among various prenyltransferases. Using these primers invarious combinations, PCR is performed with genomic DNA from the soilbacterium Paracoccus denitrificans (hereinafter referred to as “=P.denitrificans”) as a template. The gene of the present invention can beobtained by screening the genomic DNA using one of the amplified partialsequences as a probe.

1. Cloning of a Gene Coding for a Prenyl Diphosphate Synthetase

(1) Preparation of Genomic DNA

First, genomic DNA is prepared from cultures cells of a prenyldiphosphate synthetase producing bacterium such as the soil bacterium P.denitrificans.

The preparation of genomic DNA may be performed by any of theconventional methods. For example, genomic DNA can be prepared easily bythe following procedures. The above bacterium is inoculated into amedium containing 2 g of yeast extract, 10 g of Polypeptone, 1 g ofMgSO₄. 7H₂O and 1 liter of distilled water (802 medium) and cultured at30° C. for one to several days (until saturation); subsequently,bacterial cells are treated with lysozyme and further treated with asurfactant such as sodium lauryl sulfate; thereafter, proteins areremoved therefrom with an organic solvent such as phenol, chloroform orether; then, genomic DNA is precipitated with ethanol.

Subsequently, a genomic DNA library is prepared by ligating theresultant genomic DNA to a vector plasmid. This preparation may beperformed by conventional methods. For example, genomic DNA strand andplasmid DNA strand are cut with an appropriate restriction enzyme (e.g.,EcoRI, BamHI, Hind III, Sau3AI, MboI, PstI); then, these strands arejust treated with a DNA ligase (e.g., T4 DNA ligase), or they aretreated with a DNA ligase after treatment with a terminal transferase orDNA polymerase depending on the states of the resultant fragment ends,to thereby ligate DNA strands (Molecular Cloning, Cold Spring HarborLaboratory, 269, 1982; Nelson, T. et al. Methods in Enzymol., 68, 41,1979). As a vector useful for this purpose, λ phage vectors (e.g.,λgt10, Charon 4A, EMBL-3), plasmid vectors (e.g., pBR322, pSC101, pUC19,pUC119, pACYC117) or like may be enumerated. After incorporation of theabove DNA fragment into such a vector, Escherichia coli(e.g., DH1,HB101, JM109, C600, MV1184, TH2) is transformed with the vector toobtain a genomic DNA library.

(2) Preparation of Probes for Screening

First, probes to be used for screening the above genomic DNA library byhybridization are prepared. For the preparation of probes which arehighly specific to a DNA of interest, it is considered appropriate toprepare oligonucleotides coding for the regions with highly preservedamino acid residues among various organism species. These probes can beobtained by conventional chemical synthesis. As amino acid sequenceswhich satisfy the above conditions, the following preserved amino acidsequences are selected based on FIG. 3.

The sequence “(Gly or Glu) Gly Lys Arg Ile Arg Pro” (SEQ ID NO: 1) inRegion I

The sequences “(Thr or Met) Ala (Ser or Thr) Leu (Val, Ile or Leu) HisAsp” (SEQ ID NO: 4), “Ala Ser Leu Leu His Asp Asp” (SEQ ID NO: 5) and“Ala Asp Leu Arg Arg Gly” (SEQ ID NO: 6) in Region II

The sequence “Leu Ala Gly Asp Phe Leu Leu” (SEQ ID NO: 7) in Region III

The sequence “Gly Glu Leu Gln Leu” (SEQ ID NO: 8) in Region IV

The sequence “Lys Thr Ala Leu Leu Ile” (SEQ ID NO: 9) in Region V

The sequences “Phe Gln Leu Ile Asp Asp” (SEQ ID NO: 10), “Asp Asp IleLeu Asp Phe” (SEQ ID NO: 11), “Gly Lys Asn Val Gly Asp Asp” (SEQ ID NO:12) and “Asp Asp (Leu, Ile or Met) Leu Asp (Tyr or Phe) (Asn or Thr)”(SEQ ID NO: 13) in Region VI.

Regions, I, II, III, IV, V and VI correspond to amino acid positionsfrom 43 to 53, from 74 to 95, from 110 to 119, from 145 to 150, from 170to 175 and from 204 to 230, respectively, of the amino acid sequence fora Bacillus stearothermophylus-derived heptaprenyl diphosphate synthetasedisclosed in Koike- Takeshita, A. et al., (1995) J. Biol. Chem.270:18396-18400.

Examination of preserved amino acid sequences in various organismspecies can be performed among known prenyl diphosphate synthetases,such as FPS synthetases from Bacillus stearothermophylus, Escherichiacoli, Saccharomyces cerevisiae, rat and human; GGPS synthetases fromErwinia herbicola and Erwinia uredovora; and HexPS synthetase fromSaccharomyces cerevisiae.

Based on the amino acid sequences thus selected, the oligonucleotideprobes shown below are prepared.

Briefly, in the present invention, the following 12 degenerate primersare designed based on highy preserved amino acid sequences among variousprenyltransferases and on those sequences which are peculiar to medium-or long-chain prenyltransferases such as hexaprenyl diphosphate (HexPP,C30) synthetase, heptaprenyl diphosphate (Hepp, C35) (Koike-Takeshita,A. et al. (1995) J. Biol. Chem. 270:18396-18400) synthetase andoctaprenyl diphosphate (OctPP, C40) (Okada, K., J. Bacteriol. 179,3058-3060 (1997) synthetase.

Sense primers:

S1 (designed based on SEQ ID NO: 3):

5′-(CG) (AG)CGG(AT)AA(AG)C(AG) (CGT)AT(CGT)CGTCC-3′(SEQ ID NO: 14)

S2 (designed based on SEQ ID NO: 4):

5′-A(CT)(ACGT)GC(GT)(AT)C(ACGT)CT(ACGT)(CGT)T(ACGT)CACGA-3′ (SEQ ID NO:15)

S3 (designed based on SEQ ID NO: 3):

5′-GG(ACGT)GG(ACGT)AA(AG)CG(ACGT)AT(ACT)CG(ACGT)CC-3′ (SEQ ID NO: 16)

S4 (designed based on SEQ ID NO: 5):

5′-GC(ACGT)TC(ACGT)CT(ACGT)CT(ACGT)CA(CT)GACGA-3′ (SEQ ID NO: 17)

S5 (designed based on SEQ ID NO: 6):

5′-GC(ACGT)GA(CT)TT(AG)(AC)G(ACGT)(AC)G(ACGT)GG-3′ (SEQ ID NO: 18)

S6 (designed based on SEQ ID NO: 7):

5′-(CT)T(ACGT)GC(ACGT)GG(ACGT)GA(CT)TT(CT)TT(AG)TT-3′ (SEQ ID NO: 19)

Antisense primers:

A1 (designed based on SEQ ID NO: 13):

5′-(GT)T(AG)(AT)AATCGAG(TA)A(ACT)(AG)TC(AG)TC-3′ (SEQ ID NO: 20)

A2 (designed based on SEQ ID NO: 8):

5′-(ACGT)A(AG)(CT)TG(CT)AA(ACGT)A(AG)(CT)TC(ACGT)CC-3′ (SEQ ID NO: 21)

A3 (designed based on SEQ ID NO: 9):

5′-(AGT)AT(ACGT)AG(ACGT)AG(ACGT)GC(ACGT)GT(TC)TT-3′ (SEQ ID NO: 22)

A4 (designed based on SEQ ID NO: 10):

5′-(AG)TC(AG)TC(AGT)AT(CT)AA(CT)TG(AG)AA-3′ (SEQ ID NO: 23)

A5 (designed based on SEQ ID NO: 11):

5′-(AG)AA(AG)TC(ACGT)A(AG)(AGT)AT(AG)TC(AG)TC-3′ (SEQ ID NO: 24)

A6 (designed based on SEQ ID NO: 12):

5′-(AG)TC(AG)TC(ACGT)CC(ACGT)AC(AG)TT(CT)TT(ACGT)CC-3′ (SEQ ID NO: 25)

(3) Cloning of a Part of a Prenyl Diphosphate Synthetase Gene

The screening of P. denitrificans genomic DNA for the gene of thepresent invention can be performed by conventional methods such asSouthern hybridization, colony hybridization, PCR or a combination ofthese methods.

For example, genomic DNA from P. denitrificans is subjected to PCR usinga combination of the primers described above to thereby amplify a DNAfragment containing a part of a prenyl diphosphate synthetase gene. Thefragment which is believed to contain a part of the target gene isseparated by electrophoresis and recovered. After ligation of the DNAfragment to a vector, E. coli is transformed with the vector, and theDNA fragment is cloned. The thus obtained DNA fragment (pCR14) issuitable as a probe for obtaining a full length prenyl diphosphatesynthetase gene.

(4) Cloning of a Full Length Prenyl Diphosphate Synthetase Gene

As described above, probe pCR14 is a DNA fragment containing a part ofthe prenyl diphosphate synthetase gene of P. denitrificans. Thus, thescreening for a gene encoding the peptide of the prenyl diphosphatesynthetase of the invention is performed, for example, as describedbelow using pCR14.

The genomic DNA partially digested with Sau3AI is electrophoresed.Resultant DNA fragments of 5-10 kbp are extracted from the agarose geland inserted into the BamHI site of pUC119. With this plasmid, E. coliJM109 is transformed to prepare a DNA library. Then, colonyhybridization is performed with pCR14 as a probe.

(5) Determination of the Base Sequence

Each of the clones thus obtained is digested with an appropriaterestriction enzyme, followed by agarose gel electrophoresis. From themigration pattern and distance, a restriction map is prepared. Based onthis map, deletion of the DNA fragment (i.e., to make the DNA fragmentshorter) is carried out to thereby obtain a minimum clone exhibitingactivity. Then, the base sequence for the activity-exhibiting DNA isanalyzed.

The base sequence may be determined using two plasmids which contain thesame insert DNA truncated at one end in opposite directions.

The screened clone is digested with an appropriate restriction enzyme(such as EcoRI, PstI) and cloned into a plasmid (such as pUC119, pUC19).Then, the base sequence for the DNA of interest can be determined byconventional base sequence analysis methods, for example, the dideoxymethod by Sanger et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA(1977) 74:5463). The determination of the base sequence may be performedwith an automatic base sequence analyzer such as T7 Sequencing Kit(Pharmacia).

(6) Identification of the Gene

A region which is expected to be a prenyl diphosphate synthetase gene isintegrated into an expression vector, with which E. coli is transformed.The transformant is cultured and resultant cells are crushed to obtain acrude enzyme extract. By determining the activity of this extract, theprenyl diphosphate synthetase, particularly, decaprenyl diphosphatesynthetase of the invention can be identified. Also, by determining thelength of the ubiquinone side chain of the transformant, the gene can beidentified.

The base sequence for the gene coding for the prenyltransferase of theinvention is shown in SEQ ID NO: 1. The amino acid sequence for theprenyltransferase of the invention is shown in SEQ ID NO: 2. However,the amino acid sequence of SEQ ID NO: 2 may have a mutation such asdeletion, substitution or addition of at least one amino acid(preferably, one or several amino acids) as long as it can exhibitprenyltransferase activity. In addition to the base sequence shown inSEQ ID NO: 1, a base sequence which codes for the same polypeptide andwhich is only different from SEQ ID NO: 1in a degenerate codon(s) isalso. included in the gene of the present invention.

Introduction of the above mutation can be performed easily byconventional methods such as the method of Kunkel (Kunkel, T. A., Proc.Natl. Acad. Sci. (1985) 82:488).

Once the base sequence has been thus determined, the target gene can beobtained by hybridization with a DNA fragment prepared by chemicalsynthesis or PCR.

2. Preparation of a Recombinant Vector and a Transformant

A recombinant vector of the invention can be obtained by integrating thegene of the invention into an appropriate vector. A transformant of theinvention can be obtained by introducing the recombinant vector into ahost which is compatible with the initial vector.

A purified gene is inserted into a restriction site or multi-cloningsite of a suitable vector DNA to obtain a recombinant vector. With thisvector, a host is transformed.

A vector DNA into which a DNA fragment is inserted is not particularlylimited as long as it is replicable in a host cell. For example, aplasmid DNA or phage DNA may be used. As a plasmid DNA, plasmid pUC118(Takara Shuzo), plasmid pUC119 (Takara Shuzo), pBluescript SK+(Stratagene), pGEM-T (Promega) or the like may be enumerated. As a phageDNA, M13mp18, M13mp19 or the like may be enumerated.

As a host, any host may be used as long as it can express the gene ofinterest. Either an eukaryotic or prokaryotic cell may be used. Forexample, bacteria such as Escherichia coli, Bacillus subtilis; yeastsuch as Saccharomyces cerevisiae; and animal cells such as COS cells,CHO cells, etc. may be enumerated.

When a bacterium such as E. coli is used as a host, preferably therecombinant vector of the invention is capable of autonomous replicationin the host and yet has a constitution comprising a promoter, the geneof the invention and a transcription terminator sequence. Specificexamples of such E. coli include XL1-Blue (Stratagene) and JM109 (TakaraShuzo). Specific examples of an expression vector include pTrc99A andpET expression systems. As a promoter, any promoter may be used as longas it can express the gene of interest in the host such as E. coli.Specific examples of the promoter include E. coli- or phage-derivedpromoters such as trp promoter, lac promoter, PL promoter and PRpromoter. In the present invention, the transformation of E. coli can beperformed, for example, by the method of Hanahan (Hanahan, D., J. Mol.Biol. (1983) 166:557).

When yeast is used as a host, an expression vector such as YEp13 orYCp50 may be used. As a promoter, gal 1 promoter or gal 10 promoter maybe used, for example. As a method for introducing a recombinant vectorinto yeast, electroporation (Becker, D. M. Methods. Enzymol. (1991)194:182-187), the spheroplast method (Hinnen, A., Proc. Natl. Acad. Sci.USA (1978) 75:1929-1933), the lithium acetate method (Ito, H., J.Bacteriol. (1983) 153:163-168) or the like may be enumerated.

When an animal cell is used as a host, an expression vector such aspSG5, pREP4 or pZeoSV may be used. As a method for introducing arecombinant DNA into an animal cell, electroporation, the calciumphosphate precipitation method, or the like may be enumerated.

When a plasmid DNA is used as a vector DNA, if an EcoRI DNA fragment isto be inserted thereinto for example, the plasmid DNA is predigestedwith the restriction enzyme EcoRI before the insertion. Then, the DNAfragment and the digested vector DNA are mixed. The resultant mixture istreated with, for example, T4 DNA ligase (Takara Shuzo) to obtain arecombinant vector.

3. Production of the Prenyltransferase

The prenyltransferase of the invention can be produced by culturing atransformant carrying the recombinant vector obtained above. The culturemethod may be the conventional solid culture, but preferably the liquidculture is employed.

As a medium for culturing the transformant, a medium containing at leastone nitrogen source selected from yeast extract, Peptone and meatextract; at least one inorganic salt such as dipotassiumhydrogenphosphate, magnesium sulfate or ferric chloride; and, ifnecessary, sugar materials, antibiotics and vitamins may be used, forexample. If necessary, IPTG or the like may be added to the medium toinduce the expression of the gene. The pH of the medium at the start ofculture is adjusted to 6.8-7.5. The culture is conducted usually at28-42° C., preferably at around 37° C., for 5 hours to overnight.Aeration agitation culture, shaking culture, or the like may beemployed.

After completion of the culture, the prenyltransferase of the inventionmay be recovered by conventional protein purification techniques.

Briefly, cells are crushed by lysis treatment with an enzyme such aslysozyme, sonication, grinding treatment or the like to release theprenyltransferase outside the cells. Then, insoluble materials areremoved by filtration, centrifugation or the like to thereby obtain acrude polypeptide solution.

For further purification of the peptide from the above crude polypeptidesolution, conventional protein purification methods may be used. Forexample, ammonium sulfate fractionation, ion exchange chromatography,hydrophobic chromatography, gel filtration chromatography, affinitychromatography and electrophoresis may be used independently or incombination.

4. Production of Ubiquinone-10

Ubiquinones are known as a constituent of the electron transport systemin a number of organisms. The length of their isoprenoid side chainsvaries with organism species. E.coli ubiquinone has an isoprenoid sidechain of 8 isoprene units supplied by oPP synthetase; the ubiquinone ofbudding yeast Saccharomyces cerevisiae has a side chain of 6 isopreneunits; and human ubiquinone has a side chain of 10 isoprene units.

Generally, E.coli ubiquinone does not have an isoprenoid side chain of10 isoprene units. However, a ubiquinone with an isoprenoid side chainof 10 isoprene units (ubiquinone-10) can be obtained from a transformantE.coli into which the gene of the invention has been introduced. Bycrushing the transformant E.coli by sonication or the like, extractingthe cell components with hexane and finally applying them to HPLC,ubiquinone-10 can be obtained.

PREFERRED EMBODIMENTS OF THE INVENTION

Hereinbelow, the present invention will be described in more detail withreference to the following Examples, which should not be construed aslimiting the technical scope of the invention.

EXAMPLE 1 Cloning of a Prenyltransferase Gene

It is known that prenyltransferases have 7 regions which have beenhighly preserved beyond species. Then, the present inventor designeddegenerate oligonucleotides based on those highly preserved amino acidsequences among various transferases. Using these oligonucleotides asprimers in various combinations, PCR was performed with genomic DNA fromP. denitrificans as a template. Using the amplified partial sequence asa probe, screening was conducted to clone a prenyltransferase gene.

The restriction enzymes and other DNA modification enzymes used in thecloning were obtained from Takara Shuzo, Toyobo and New England BioLabs.

(1) Preparation of Genomic DNA from P. denitrificans and Creation of aGenomic Library

P. denitrificans was inoculated into 100 ml of 802 medium (10 g ofPolypeptone, 2 g of yeast extract, 1 g of MgSO₄. 7H₂O, 1 liter ofdistilled water, pH 7.0) and cultured at 30° C. until saturation. Then,cells were harvested, and genomic DNA was prepared according to themethod of Saito et al. (Biochim. Biophys. Acta 72, 619-629 (1963))

P. denitrificans was obtained from American Type Culture Collection(ATCC14907).

The genomic DNA from P. denitrificans was partially digested with arestriction enzyme. DNA fragments of a specific length were recovered.Then, a library was prepared. By these procedures, screening efficiencyis improved compared to the screening of an entire genomic library.

Briefly, 1 U of Sau3A1 was added to 50 μg of the genomic DNA. Theresultant mixture was incubated at 37° C. A specific amount of samplewas taken in every 5 minutes from the start of the incubation up to 90minutes. Then, the reaction was terminated. Each sample waselectrophoresed on 0.8% agarose gel. Fragments of 5-10 kbp wererecovered from the gel and ligated to pUC119-BamHI vector individually.With this vector, E. coli DH5α was transformed. The resultanttransformants were cultured in LB medium to prepare glycerol stockshaving a glycerol concentration of 30%. Thus, 10 libraries eachcontaining about 2000 clones were prepared. From these libraries,plasmid DNAs were recovered.

(2) Design of PCR Primers

In the present invention, the following 12 degenerate primers weredesigned based on highly preserved amino acid sequences among variousprenyltransferases, particularly on those sequences which are peculiarto medium- or long-chain prenyltransferases such as hexaprenyldiphosphate (HexPP, C30) synthetase, heptaprenyl diphosphate (HepPP,C35) (Koike-Takeshita, A. et al. (1995) J. Biol. Chem. 270:18396-18400)synthetase and octaprenyl diphosphate (OctPP, C40) (Okada, K. et al., J.Bacteriol. 179, 3058-3060 (1997)) synthetase.

Sense primers:

S1 (designed based on SEQ ID NO: 3):

5′-(CG)(AG)CGG(AT)AA(AG)C(AG)(CGT)AT(CGT)CGTCC-3′(SEQ ID NO: 14)

S2 (designed based on SEQ ID NO: 4):

5′-A(CT)(ACGT)GC(GT)(AT)C(ACGT)CT(ACGT)(CGT)T(ACGT)CACGA-3′ (SEQ ID NO:15)

S3 (designed based on SEQ ID NO: 3):

5′-GG(ACGT)GG(ACGT)AA(AG)CG(ACGT)AT(ACT)CG(ACGT)CC-3′ (SEQ ID NO: 16)

S4 (designed based on SEQ ID NO: 5):

5′-GC(ACGT)TC(ACGT)CT(ACGT)CT(ACGT)CA(CT)GACGA-3′ (SEQ ID NO: 17)

S5 (designed based on SEQ ID NO: 6):

5′-GC(ACGT)GA(CT)TT(AG)(AC)G(ACGT)(AC)G(ACGT)GG-3′ (SEQ ID NO: 18)

S6 (designed based on SEQ ID NO: 7):

5′-(CT)T(ACGT)GC(ACGT)GG(ACGT)GA(CT)TT(CT)TT(AG)TT-3′ (SEQ ID NO: 19)

Antisense primers:

A1 (designed based on SEQ ID NO: 13):

5′-(GT)T(AG)(AT)AATCGAG(TA)A(ACT)(AG)TC(AG)TC-3′ (SEQ ID NO: 20)

A2 (designed based on SEQ ID NO: 8):

5′-(ACGT)A(AG)(CT)TG(CT)AA(ACGT)A(AG)(CT)TC(ACGT)CC-3′ (SEQ ID NO: 21)

A3 (designed based on SEQ ID NO: 9):

5′-(AGT)AT(ACGT)AG(ACGT)AG(ACGT)GC(ACGT)GT(TC)TT-3′ (SEQ ID NO: 22)

A4 (designed based on SEQ ID NO: 10):

5′-(AG)TC(AG)TC(AGT)AT(CT)AA(CT)TG(AG)AA-3′ (SEQ ID NO: 23)

A5 (designed based on SEQ ID NO: 11):

5′-(AG)AA(AG)TC(ACGT)A(AG)(AGT)AT(AG)TC(AG)TC-3′ (SEQ ID NO: 24)

A6 (designed based on SEQ ID NO: 12):

5′-(AG)TC(AG)TC(ACGT)CC(ACGT)AC(AG)TT(CT)TT(ACGT)CC-3′ (SEQ ID NO: 25)

(3) Amplification of a Prenyltransferase Gene Fragment by PCR

A PCR was conducted using TaKaRa Taq from Takara Shuzo. Usually, thecomposition of the reaction mixture was as follows. As a template, thegenomic DNA from P. denitrificans was used.

TaKaRa Taq 2.5 U Tris-HCl (pH 8.3) 10 mM KCl 50 mM MgCl₂ 1.5 mM dNTPmixture 0.2 mM each Template 0.1 μg Primer 1 (any one of SEQ ID NOS:14-19) 2.5 μg Primer 2 (any one of SEQ ID NOS: 20-25) 2.5 μg H₂O to make100 μl

The PCR was conducted with DNA Thermal Cycler PJ2000 (Takara Shuzo). ThePCR cycles were as described below.

Briefly, 5 cycles of denaturation at 97° C. for 30 seconds, annealing at40° C. for 30 seconds, and extension at 70° C. for 1 minute; then 30cycles of denaturation at 97° C. for 30 seconds, annealing at 55° C. for30 seconds, and extension at 70° C. for 1 minute were carried out.

After completion of the PCR, the products were subjected toelectrophoresis with 1×TBE/5% acrylamide gel. The amplified DNAfragments were confirmed (FIG. 4) and recovered by the gel recoverymethod. The DNA clone obtained by the reaction using primers S4 (SEQ IDNO: 17) and A6 (SEQ ID NO:25) was designated “pCR14”. Then, pCR14 waspurified and sub-cloned into pT7BlueT-vector (Novagen). The basesequence of pCR14 was determined with an automatic base sequenceanalyzer (ABIPRISM™ 310 Genetic Analyzer), followed by analysis using agene analysis software GENETIX for comparison with the amino acidsequences of other prenyltransferases.

As a result, the amino acid sequence encoded by pCR14exhibited 45.7%homology to the amino acid sequence of E. coli OctPP synthetase, 35.5%homology to the amino acid sequence of B. stearothermophilus HepPPsynthetase, and 31.8% homology to the amino acid sequence of E.coli FPPsynthetase (FIG. 5).

(4) Southern Blot Analysis

For the sub-cloned and sequenced PCR product (i.e., pCR14), PCR primers(BS and BA; FIG. 6) were newly designed based on its sequence locatedinside of the above-described degenerate oligo primers (S4 and A6).Using these primers, a DNA fragment to be used as a probe forhybridization was amplified by PCR and recovered.

Sense primer

BS: 5′-CCGGCCGACGCAAACCTT-3′ (SEQ ID NO: 26)

Antisense primer

BA: 5′-CTGCTGCACCGCCGGGTC-3′ (SEQ ID NO: 27)

The amplification was conducted 30 cycles, 1 cycle consisting ofdenaturation at 97° C. for 30 seconds, annealing at 55° C. for 30seconds and extension at 72° C. for 1 minute.

Using a 300 bp fragment thus amplified as a probe, Southern blotanalysis of the genomic DNA from P. denitrificans was performed. The PCRproduct was labelled with [α-³⁵S]dCTP (Amersham) using a commercial kit(Ready To Go DNA Labelling Beads; Pharmacia). The labelling wasperformed according to the protocol attached to the kit.

A filter was prepared by the following procedures. The chromosomal DNAfrom P. denitrificans (10 μg) was completely digested with ApaI, EcoRIand BamHI separately. Each of these digests was electrophoresed on0.5×TBE/0.7% agarose gel, alkali denatured, and then transferred to anitrocellulose membrane filter (Zeta Probe Blotting Membrane from BioRador Hybond-N+ from Amersham).

The composition of a hybridization solution was varied as shown belowdepending on homology to the probe. The filter was incubated in thesolution at a constant temperature of 42° C. to perform prehybridizationand hybridization.

(i) Stringent conditions (homology=approx. 100%)

5×SSC

5×Denhardt's solution

1% SDS

0.2 mg/ml denatured salmon sperm DNA

50% formamide

³⁵S-probe (this is omitted in the prehybridization)

(ii) Moderate conditions (homology=approx. 50%)

5×SSC

5×Denhardt's solution

1% SDS

0.2 mg/ml denatured salmon sperm DNA

25% formamide

³⁵S-probe (this is omitted in the prehybridization)

Conditions for washing after the hybridization were also varied asfollows.

Stringent conditions (homology=approx. 100%):

0.1% SDS, 0.1×SSC, at 68° C.

Moderate conditions (homology=approx. 50%):

0.1% SDS, 2×SSC, at 55° C.

After washing, the filter was exposed to a Fuji imaging plate andanalyzed with Fuji BAS-2000 Bioimage Analyzer System.

As a result, under the stringent conditions (under which the homologybetween the detected bands and the primers would be approximately 100%),a 16.5 kbp band was detected when the genomic DNA had been digested withApaI; a 18.5 kbp band was detected when the genomic DNA had beendigested with EcoRI; a 11.2 kbp band and a slightly weakly hybridizing4.2 kbp band were detected when the genomic DNA had been digested withBamHI (FIG. 7A). The slightly weak 4.2 kbp band is predicted to containa sequence which is highly homologous to the sequences obtained thistime that appear to code for a prenyltransferase gene. In other words,this 4.2 kbp band is predicted to contain another prenyltransferase gene(FPP synthetase) of P. denitrificans.

On the other hand, under moderate conditions (under which the homologybetween the detected bands and the primers would be approximately 50%),additional bands were confirmed as follows: a 7.4 kbp band when thegenomic DNA had been digested with ApaI, a 5.3 kbp band when the genomicDNA had been digested with EcoRI and a 5.2 kbp band when the genomic DNAhad been digested with BamHI (FIG. 7B). These bands are very likely tocontain other prenyltransferase genes.

(5) Recovery of a Full Length Gene by Colony Hybridization

In order to recover a full length gene containing the gene fragmentamplified by PCR, colony hybridization was conducted using the sameprobe as used in the Southern hybridization. First, the genomic DNA fromP. denitrificans was partially digested with Sau3AI. Then, 5-10 kbpfragments were recovered and sub-cloned into pUC119-BamHI vector tothereby obtain 10 libraries separately each of which contained about2000 clones. Plasmid was recovered from each library, digested withEcoRI and then subjected to Southern hybridization. Thus, thoselibraries which surely contained the gene of interest were selected.

As a result, strongly hybridizing bands were detected in libraries Nos.9 and 10 among the 10 libraries (Nos. 1-10) (FIG. 8).

Then, library No. 10 which had exhibited the strongest bands in Southernhybridization was subjected to colony hybridization to thereby obtain 3positive clones. Plasmids were recovered from them and designated p11A1,p11A2 and p11C1, respectively. Since each of these clones had an insertof about 7 kbp, it was confirmed by PCR if these clones contained thegene of interest.

Briefly, using PCR primers BS and BA described above, a PCR wasperformed with these clones and pCR14 as templates. It was observedwhether a band similar to that amplified in pCR14 is also amplified inthese clones (FIG. 9). The PCR was performed 25 cycles, 1 cycleconsisting of denaturation at 98° C. for 30 seconds, annealing at 67° C.for 30 seconds and extension at 74° C. for 30 seconds.

As a result, only p11A1 (lane 1) exhibited amplification of a DNAfragment of about 300 bp similar to the fragment amplified in pCR14(lane 4) (FIG. 9). No amplification was recognized in p11A2 and p11C1under these conditions. Therefore, it is believed that they do notcontain a full length gene of interest or they contain a differentprenyltransferase gene.

Subsequently, by preparing a restriction map for p11A1, it wasascertained where the sequence identical with pCR14 is contained in itsinsert of about 7 kbp (FIG. 10). Also, it was confirmed that a fulllength prenyltransferase gene was contained (FIG. 10). As a result, itwas found that the sequence identical with pCR14 is located about1.1-1.5 kbp from an end of the insert of p11A1. Considering that theaverage gene length of prenyltransferases is about 1 kbp and that thepreserved Regions II to VI are contained in pCR14, the insert of p11A1was expected to contain a full length of a prenyltransferase gene.

(6) Deletion of p11A1 and Determination of the Base Sequence

First, the present inventor decided to determine the total base sequencefor the prenyltransferase gene contained in p11A1. Deletion of p11A1 wasallowed to proceed from the BglII site located 4 kbp downstream of thesequence identical with pCR14, and finally, DNA fragments were cut outat the BamHI site located 430 bp upstream of pCR14. The resultantfragments were ligated to pUC119 vector digested with SmaI and BamHI.

The vectors were screened by colony hybridization. The recovered clonewas cut with BglII and then digested from the 3′ end with exonucleaseIII. The reaction was terminated after an appropriate time period.Thereafter, resultant DNA fragments were blunt-ended with mung beannuclease or Klenow fragment. Finally, the DNA fragments were cut out bydigesting with BamHI.

These fragments were electrophoresed on 3.5% acrylamide gel. Thereafter,the fragments were recovered from the gel and used to transform E. coliDH5α. Several single transformants were selected and plasmids wererecovered therefrom.

These plasmids carrying a deletion product were applied to a sequencer(from ABI) to thereby determine the base sequence for the full lengthgene.

As a result, an ORF was found out which contains a base sequenceidentical with pCR14 and has in its amino acid primary sequence the 7preserved areas peculiar to prenyltransferases (FIG. 11; SEQ ID NO: 28).

This ORF has 4 ATG codons which may be the translation initiation point.Of these, the third methionine which is close to Shine-Dalgarnoconsensus sequence and has a reasonable distance from it is believed tobe the translation initiation point.

The amino acid primary sequence of this ORF was compared with theprimary sequences of major prenyltransferases so far cloned. As aresult, this ORF has 34.9% homology to E. coli FPP synthetase; 31.1%homology to B. stearothermophilus FPP synthetase; 31.8% homology to E.uredovora GGPP synthetase; 26.3% homology to M. luteus BP-26 HexPPsynthetase; 34.4% homology to B. stearothermophilus HepPP synthetase;and 44.2% homology to E. coli OctPP synthetase (FIG. 12).

During the process of deletion, a downstream base sequence of about 1kbp adjacent to the ORF of the prenyltransferase contained in p11A1 wasdetermined.

As a result, a typical terminator sequence characterized by a repetitivesequence and repetition of T was found at 25 bp downstream of the ORFtermination codon TGA (nucleotide positions from 1174 to 1201 in FIG. 11and SEQ ID NO: 28). Therefore, it was found that there is no ORF formingan operon in the downstream of this prenyltransferase gene.

Also, an upstream base sequence of about 1 kbp adjacent to the BamHIsite upstream of the ORF was determined. As a result, it was found thatan operon of 3.3 kpb exists in the upstream of the prenyltransferasegene, which operon is composed of the β-ketothiolase gene and acetyl-CoAreductase gene of P. denitrificans already cloned and analyzed(Yabuntani, T. et al., (1995) FEMS Microbial. Lett. 133:85-90). Althoughthese two genes are forming an operon, the operon is terminated by aterminator. Thus, they are not forming an operon with the gene of theinvention (FIG. 13).

EXAMPLE 2 Construction of a High Expression System for thePrenyltransferase

In the present invention, a system which allows compulsive expression ofthe prenyltransferase with a strong trc promoter and SD sequence frompTrc99A was constructed by introducing an NcoI site into the initiationcodon (ATG) of the ORF and then sub-cloning it into the NcoI site of ahigh expression vector pTrc99A.

An expression plasmid was prepared by introducing into a plasmid an ORFwhich starts from an ATG methionine codon located at around thepredicted position for the initiation codon based on the 7 preservedregions of known prenyltransferases.

(1) Preparation of a High Expression Plasmid

Of those ORFs which were believed to be a prenyltransferase as a resultof the confirmation of base sequences, the ORF in which the thirdmethionine is the initiation codon was introduced into the NcoI-BamHIsite of the expression vector pTrc99A.

First, restriction sites were introduced into the ORF by PCR usingvariant oligoprimers so that the ORF could be introduced into thevector. An NcoI site (CCATGG) was introduced into the Met codon (ATG)which is the translation initiation point. Also, a BamHI site (GGATCC)was introduced into 84bp downstream of the termination codon TGA. In theintroduction, primers were designed in such a manner that the amino acidimmediately after the initiation codon was not changed. The sequences ofthe PCR primers for introducing restriction sites are as follows.

    Sense primer (SEQ ID NO: 29) DP03:5′-ATCGCCCATGGGCATGAACGAAAACGTCTC-3′               NcoI     Antisenseprimer (SEQ ID NO: 30) DP13: 5′-GAGGGATCCTATAACAACTGAGGCAGCG-3′            BamHI

By performing a PCR with these primers, a gene fragment having arestriction site at each end was amplified. As a polymerase for use inthe PCR, KOD DNA polymerase from Toyobo was employed which is reportedto be superior to Taq DNA polymerase and Pfu DNA polymerase in accuracyin DNA synthesis and amplification efficiency (Barnes, W. M. (1994)Proc. Natl. Acad. Sci. USA 91:2216-2220). The composition of thereaction mixture and the PCR cycles are as described below.

KOD DNA polymerase 2.5 U Tris-HCl (pH 8.3) 120 mM KCl 10 mM (NH₄)₂SO₄ 6mM MgCl₂ 1 mM dNTP mixture 0.2 mM each Template 0.1 μg Primer (DPO3)1.25 μM Primer (DP13) 1.25 μM H₂O to make 50 μl

The PCR was conducted 25 cycles, 1 cycle consisting of denaturation at98° C. for 30 seconds, annealing at 67° C. for 30 seconds and extensionat 74° C. for 30 seconds.

After completion of the PCR, the products were digested with NcoI andBamHI, electrophoresed on 0.8% agarose gel and recovered. The resultantNcoI-BamHI gene fragment was sub-cloned into the NcoI-BamHI site ofpTrc99A (Amann, E. et al. (1988) Gene 69:301-305). The thus obtainedexpression plasmid was designated pDPm3. This plasmid was also sequencedto confirm the sequence of the vector and the junction sites.

As a result, it was confirmed that the ORF of the prenyltransferase issurely inserted into this plasmid and ligated without frameshift to theNcoI site.

Thereafter, E. coli DH5α was transformed with this expression plasmidpDPm3.

The E. coli carrying the expression plasmid pDPm3 (pDPm3/DH5α) has beendeposited under the terms of the Budapest Treaty at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (1-3 Higashi 1-chome, Tsukuba City, IbarakiPref., Japan) on Jul. 9, 1997 under Accession No. FERM BP-6259.

(2) High Expression of the Prenyltransferase in E. coli

The E. coli transformed with the expression plasmid pDPm3 was inoculatedinto LB medium (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1%glucose) containing 50 μg/ml ampicillin and cultured overnight at 37° C.Subsequently, 1 ml of this culture fluid was inoculated into 100 ml ofM9 nutrient medium (0.2% M9 salt, 0.2% glycerol, 0.2% yeast extract)containing 50 μg/ml ampicillin and cultured at 30° C. When the turbidityreached at A₆₀₀=0.6-0.8, isopropyl β-D-thiogalactopyranoside (IPTG) wasadded thereto to give a final concentration of 1 mM. Then, the cellswere cultured overnight at 30 ° C.

The culture fluid was centrifuged at 4° C. at 1,000×g for 10 minutes andwashed with 50 mM potassium phosphate buffer (pH 7.2). The resultantcells were suspended in a lysis buffer (50 mM potassium phosphate buffer(pH 7.2), 5 mM EDTA, 1 mM β-mercaptoethanol, 1 mM PMSF) and subjected tosonication [(ultrasound 10 sec.+ice cooling 2 min.)×10 cycles], tothereby disrupt cells. The sonication was conducted with Sonifier 250from Branson. After disruption, the cell suspension was centrifuged at4° C. at 15,000×g for 30 minutes, and the supernatant was collected as acrude enzyme extract.

Subsequently, prenyltransferase activity was determined as describedbelow.

A 200 μl reaction solution shown below was prepared using an appropriateamount of the crude enzyme extract, various allylic primers and[¹⁻¹⁴C]IPP (54 or 57 Ci/mol; Amersham).

Potassium phosphate buffer (pH 7.2) 50 mM MgCl₂ 5 mM β-mercaptoethanol10 mM Triton X-100 0.5% [¹⁴C] IPP (1Ci/mol) 125 μM Allylic primer 25 μMCrude Enzyme Extract Appropriate volume Total 200 μl

The solution was incubated at 37° C. for 1 hour to allow an enzymereaction. Then, 200 μl of saturated aqueous NaCl solution and 1 ml ofn-BuOH saturated with saturated aqueous NaCl solution were added theretoand agitated well. The resultant solution was centrifuged to extract thereaction products. 200 μl of the BuOH layer was collected, and 3 ml ofClear Sol was added thereto. Then, the enzyme activity was determined bymeasuring the radioactivity in the BuOH extract with a liquidscintillation counter. The enzyme activity was expressed in unit, oneunit being the amount of IPP (nmol) taken into the reaction products per1 minute.

As a result, prenyltransferase activity which is believed to be derivedfrom a foreign gene was confirmed in the IPTG-induced, pDPm3-transformedE.coli (Table 2).

TABLE 2 Enzyme Activity − (× 10⁻³ unit) + Transformant Triton X-100Triton X-100 DH5 α/pDPm3 1.27 0.553 DH5 α/pDPm3 + IPTG 9.05 54.4 DH5α/pUC119 + IPTG 1.78 1.78

It is noted that significant transferase activity was not confirmed inE.coli which was transformed with pDPm3 but not induced with IPTG. Thisindicates that the expression of this prenyltransferase activity isunder the strong control of the trc promoter.

(3) Analysis of the Reaction Product by Reversed Phase TLC

Subsequently, the prenyl diphosphate generated by the prenyltransferasewas hydrolyzed with an acid phosphatase. The resultant hydrolysate wasanalyzed by reversed phase thin layer liquid chromatography (TLC). Theacid phosphatase was purchased from Boehringer Mannheim. As a thin layerchromatography plate, LKC18 of Whatman Chemical Separation was used.

Briefly, a reaction was performed using the crude enzyme extract. Thereaction products (prenyl diphosphates) were extracted with n-butanol(n-BuOH) and hydrolyzed with an acid phosphatase into correspondingprenols in the reaction solution the composition of which is shown below(Fujii, H. et al. (1982) Biochim. Biophys. Acta. 712:716-718).

Butanol layer 0.8 ml 1 M acetate buffer (pH 4.7) 0.57 ml Methanol 1.2 mlAcid phosphatase 2 mg H₂O 0.43 ml Total 3 ml

The hydrolysis was performed overnight at 37 ° C. After completion ofthe reaction, 1 ml of saturated aqueous NaCl solution and 3 ml ofn-pentane were added thereto and agitated, to thereby extract the prenolwith the pentane. The pentane layer was recovered and washed with H₂O.Then, the pentane extract was concentrated with a centrifugal evaporatorand developed by reversed phase TLC (with LKC-18) to identify thereaction products (eluent: aceton:H₂O =19:1). The positions of variousprenols used as standard samples were visualized by exposure to iodinevapor. The TLC plate was exposed to a Fuji imaging plate, which was thenanalyzed with Fuji BAS-2000 Bioimage Analyzer to detect the positions ofradioactive prenols.

The results are shown in FIG. 14.

E. coli is known to have three prenyltransferase activities of FPPsynthetase, OPP synthetase and undecaprenyl diphosphate synthetase.However, in the E. coli transformed with pDPm3, production of decaprenyldiphosphate has been confirmed (FIG. 14). Therefore, it has become clearthat the gene of the invention is a DPP synthetase gene.

The substrate specificity of this DPP synthetase has been examined onvarious allyl primers. The unit of enzyme activity is as defined above.

As a result, this DPP synthetase exhibited the maximum activity with FPP(see Table 3; enzyme activity 54.4), though slight activity was observedwith GPP. When EEE-geranylgeranyl diphosphate (trans-GGPP) orZEE-geranylgeranyl diphosphate (cis-GGPP) was used as a substrate,strong activity was observed with trans-GGPP while little activity wasobserved with cis-GGPP. These results support that the enzyme of theinvention is an enzyme catalyzing E-type chain elongation.

TABLE 3 Substrate Specificity of the Prenyltransferase Enzyme Activity −(× 10⁻³ unit) Allylic Substrate Triton X-100 Triton X-100 DMAPP 1.634.17 GPP 1.78 19.8 EE-FPP 9.05 54.4 EEE-GGPP 3.22 35.33 ZEE-GGPP 2.473.68

(4) Confirmation of High Expression of the Prenyltransferase

High expression of the prenyltransferase by means of the expressionplasmid was confirmed as described below. Briefly, the crude enzymeextract was analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using RESEP GEL 12.5% (Wakamori K.K.). The positions of bandsstained with Coomassie Brilliant Blue R-250 were compared with molecularmarkers (SDS-PAGE Molecular Weight Standards, Broad Range; BioRad).

The procedures basically followed the method of Laemmli (Laemmli U.K.(1970) Nature 227:680-685).

As a result, a band which is believed to be due to high expression wasobserved at around 36 kDa in the precipitated fraction aftercentrifugation at 15,000×g (FIG. 15, lane 6).

(5) Analysis of the Ubiquinone Side Chain Length

Ubiquinones were extracted from the E.coli transformed with pDPm3,followed by analysis of the chain length of isoprene side chains.Ubiquinone extraction was performed as described below.

First, 0.3 g of wet cells were suspended in 2 ml of methanol-0.3% NaClsolution (10:1, v/v) (hereinafter referred to as “extraction solution”)and sonicated (30 min×4 times). Then, 1 ml of extraction solution wasadded thereto, and hexane extraction was performed twice. The extractwas washed with extraction solution to remove the hexane and thendissolved in 1 ml ethanol, followed by HPLC.

HPLC equipment from Hitachi was used. As a column, LiChrosorb RP-18 (5μm) (Merck) was used. As an eluent, EtOH (99.8%) was fed at 1 ml/min.Detection was conducted at 275 nm.

The results are shown in FIG. 16 and Table 4. In the E. coli transformedwith the vector plasmid pTrc99A alone, only ubiquinone-8 (UQ-8) wasdetected. On the other hand, in the E.coli transformed with pDPm3, about20% of the total ubiquinone was replaced with ubiquinone-10. Further, inthe E. coli transformed with pDPm3 and cultured under induction withIPTG, about 70% of the total ubiquinone was replaced with UQ-10.

TABLE 4 Ubiquinone Yield (μg/g wet cell) Cell UQ-8 UQ-10 DH5 α/pUC11972.7 — DH5 α/pDPm3 16.2 4.51 DH5 α/pDPm3 + IPTG 6.05 17.5 P.denitriticans — 256

From the above, it has been confirmed that the isolated gene is codingfor a decaprenyl diphosphate synthetase. Although E. coli does notnaturally have the ability to produce ubiquinone-10, it has becomepossible to allow E. coli to produce ubiquinone-10 by transforming thisbacterium with this gene of the invention (FIG. 17).

EFFECT OF THE INVENTION

According to the present invention, a decaprenyl diphosphate synthetase,a gene coding for the synthetase, a recombinant vector comprising thegene, a transformant transformed with the vector, a method for producinga decaprenyl diphosphate synthetase, and a method for producingubiquinone-10 with a transformed microorganism are provided. The enzymeand the gene of the present invention are useful for the production ofthe enzyme, the production of ubiquinone-10, and the like.

What is claimed is:
 1. A method for producing ubiquinone-10 comprising:(A) culturing a transformant transformed with a recombinant vectorcomprising (1) a gene coding for a recombinant protein (a) or (b)defined below: (a) a protein having the amino sequence of SEQ ID NO:2;or (b) a protein which has the amino acid sequence of SEQ ID NO:2 havingdeletion, substitution or addition of one amino acid and which hasdiphosphate synthase activity; or (2) a gene having the nucleotidesequence of SEQ ID NO:1; and (B) recovering the ubiquinone-10 from theresultant culture.
 2. A method for producing ubiquinone-10 comprising:(A) culturing a transformant transformed with a recombinant vectorcomprising a gene having the nucleotide sequence of SEQ ID NO:1; and (B)recovering the ubiquinone-10 from the resultant culture.
 3. A method ofclaim 1, wherein said recovering step comprises: (C) lysing theresultant culture; (D) extracting a crude culture lysate with a solvent;and (E) separating the ubiquinone-10 from the resultant culture lysate.4. A method of claim 2, wherein said recovering step comprises: (C)lysing the resultant culture; (D) extracting a crude culture lysate witha solvent; and (E) separating the ubiqunone-10 from the resultantculture lysate.
 5. A method of claim 1, wherein said recovering stepcomprises: extracting a ubiquinone-10 from the resultant culture with asolvent.
 6. A method of claim 2, wherein said recovering step comprises:extracting a ubiquinone-10 from the resultant culture with a solvent.